| Objective: To study the effect of Shengxue Tongbian granule on interstitial cells of cajal(ICC)in rats with slow transit constipation(STC)of blood deficiency and intestinal dryness type,To explore the intervention effect of Shengxue Tongbian granule on ICC autophagy in STC rats.Methods: After a week of adaptive feeding,45 healthy male SD rats were randomly divided into blank control group,model control group,Shengxue Tongbian granule group and positive western medicine control group.A rat model of slow transit constipation(STC)with blood deficiency and intestinal dryness was established by orally administering loperamide hydrochloride combined with tail bloodletting.The blank control group was only gavage distilled water with the same volume for 12 days,and the other three groups were gavage loperamide hydrochloride(3.0mg/kg/d)by given for 12 days,with tail bleeding at the first,sixth and twelfth days of modeling,about 2ml each time.All experimental groups were given gavage once a day.By evaluating the general conditions of mental state,activity,hair,body weight change,fecal water content and intestinal ink propulsion rate of rats in each group before and after modeling to judge that the modeling is successful.The experimental groups were divided into the same groups as before,with 8 rats left in each group.According to the equivalent dose conversion method between animals and humans,it is calculated that the raw drug dosage by gavage is 1.381g/kg/d in Shengxue Tongbian granule group and 1.293mg/kg/d in positive western medicine control group.According to the weight of rats,the administration volume is0.2ml/10 g,and 5ml/day/animal according to the average weight of rats.The blank control group and the model control group were gavage the same volume of distilled water,and all the experimental groups were given gastric lavage once a day for 14 days.After the treatment,the general conditions of mental state,activity and whether the hair is shiny were recorded before and after the treatment.Observe the effects of drugs on the body weight and fecal water content of rats,count the deaths of rats in each group,and calculate the intestinal ink propulsion rate of rats in each group after treatment.The ultrastructure of ICC was observed by transmission electron microscope,the expression level of C-kit mRNA was detected by RT-qPCR,and the expression of C-kit protein was detected by Western Blot.Results: Evaluation of modeling effect: The general situation,body weight,fecal moisture content and intestinal propulsion rate of the four groups of rats before and after modeling were comprehensively analyzed.The results showed that compared with the blank control group,the model control group,the Shengxue Tongbian granule group and the positive western medicine control group,the general situation became worse,the weight increased slowly(P<0.05),the fecal moisture content decreased(P<0.05)and the intestinal propulsion rate decreased(P<0.05).Treatment evaluation:1.General situation and weight change: After treatment,the general state of the rats in the model control group,the Shengxue Tongbian granule group and the positive western medicine control group was improved compared with that before treatment.Compared with the blank control group,the weight gain of the model control group decreased significantly before and after treatment(P<0.05).Compared with the model control group,the body weight of rats in Shengxue Tongbian granule group and positive western medicine control group increased(P<0.05),but the body weight of Shengxue Tongbian granule group increased more than that of positive western medicine control group,with statistical significance(P<0.05).2.fecal moisture content: After 2 weeks of drug intervention,the average fecal moisture content of the four groups of rats was different,and with significant statistical difference(F=339.234,P<0.05).Further pairwise comparison showed that compared with the model control group,the water content of Shengxue Tongbian granule group and positive western medicine control group increased significantly(P<0.05);However,compared with Shengxue Tongbian granule Group,the positive western medicine control group had a lower water content,with statistical difference(P<0.05).3.Intestinal propulsion rate: After 2 weeks of treatment,compared with the model control group,the intestinal propulsion rate of rats in Shengxue Tongbian granule group and positive western medicine control group increased,with statistical differences(P<0.05);After 2 weeks of treatment,there was no significant statistical difference in the intestinal propulsion rate between the positive western medicine control group and the Shengxue Tongbian granule group(P>0.05).4.Transmission electron microscope: In the blank control group,the overall structure of the colon ICC is basically normal.It has abundant cytoplasmic organelles,large nuclei and full mitochondrial structure,no obvious swelling and necrosis of mitochondria,no obvious swelling and degranulation of endoplasmic reticulum.It has close connection between ICC and surrounding smooth muscle cells,and a small number of autophagy bodies.In the model control group,the overall structure of the colon ICC was abnormal.The number of cytoplasmic organelles was significantly reduced,the nucleus was larger and the mitochondria were obviously swollen.A small amount of endoplasmic reticulum expanded and degranulated,ICC was loosely connected with nerve fibers,and autophagy bodies were obvious in the tissue.In Shengxue Tongbian granule group,the overall structure of colon ICC was slightly abnormal.With abundant cytoplasmic organelles,large nuclei,swollen mitochondria.A small amount of endoplasmic reticulum expanding and degranulation,loose connection between ICC and surrounding smooth muscle cells,obvious gap expansion and no obvious autophagy body structure.In the positive western medicine control group,the overall structure of the colon ICC was slightly abnormal.The number and structure of various organelles were basically normal,the nucleus was large,the connection between ICC and surrounding smooth muscle cells was loose,the gap was large,the connection between ICC and nerve fibers was loose,and autophagy bodies were seen in the tissue.5.RT-qPCR: Compared with the blank control group,the expression index of C-kit mRNA in the colon tissue of the model control group decreased significantly(P<0.05);Compared with the model control group,the expression level of C-kit mRNA in the colon tissue of the rats in Shengxue Tongbian granule group and positive western medicine control group increased in different degrees after treatment(P<0.05).Compared with Shengxue Tongbian granule Group,there was no statistical difference in the expression level of C-kit mRNA in colon tissue of rats in positive western medicine control group(P>0.05).6.Western Blot: Compared with the blank control group,the expression index of C-kit protein in the colon tissue of the model control group decreased significantly(P<0.05).Compared with the model control group,the expression level of C-kit protein in the colon tissue of the rats in Shengxue Tongbian granule group and positive western medicine control group increased in different degrees after treatment(P<0.05).Compared with Shengxue Tongbian granule Group,there was no statistical difference in the expression level of C-kit protein in the positive western medicine control group(P>0.05).Conclusion:1.Shengxue Tongbian granule can improve the symptoms of slow transit constipation rats with blood deficiency and intestinal dryness induced by loperamide hydrochloride by gastric perfusion combined with tail bloodletting,improve intestinal peristalsis and promote the recovery of intestinal transmission function.2.Compared with the rats of blank control group,the rats of ICC in model control group have damaged ultrastructure,have abnormal overall structure,swollen mitochondria and obvious autophagosomes,which may be related to the aggravation of ICC autophagy in rats of model control group.3.Shengxue Tongbian granule can improve intestinal motility in rats with slow transit constipation due to blood deficiency and intestinal dryness,and its mechanism may be achieved by up-regulating the expression of C-kit in rat colon tissue,inhibiting excessive autophagy of ICC in colon tissue,increasing the number of ICC and repairing its ultrastructure and function,thus affecting intestinal transit function and treating STC. |
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